Gibberellin 3β-hydroxylase genes of rice and uses thereof

ABSTRACT

Genomic DNA and cDNA encoding GA 3β-hydroxylase were isolated from rice. When the expression of these genes was suppressed in rice plants, the plants became dwarfed compared with the wild type plants.

This application claims the benefit of prior-filed Japanese Patent Application 11/361608 (filed Dec. 20, 1999) entitled “Rice-origin Gibberellin 3β-Hydroxylase Genes and Utilization Thereof”. The entire content of the above-referenced application is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to rice genes involved in gibberellin biosynthesis and uses of these genes.

BACKGROUND ART

Multicellular organisms have a large number of specialized organs and tissues that are assembled to form a functional unit. Coordination of various parts of an organism is achieved by chemical messenger substances termed hormone. Plant hormones are naturally occurring substances, effective in very small amounts that act as signals to stimulate or inhibit growth or regulate development. Nowadays, the following molecules are generally recognized to be plant hormones: auxins, gibberellins, cytokinins, abscisic acid, brassinolide, and ethylene.

In animals, hormones are usually synthesized in special glands and distributed via the bloodstream within the organism. Thus, they reach the target site and responsive tissues that are ready to react. There they trigger specific regulatory processes. This classical concept of hormones that was originally developed for animals was extended to higher plants. In many cases, plant hormones are active in specific target tissues, which are often different from the tissues in which the hormone is produced. However, all the plant hormones can also be detected in many tissues of the multicellular plant. This indicates that there is frequently no obligatory division between the site of synthesis and the site of action of plant hormones. If required, they are able to act on the same cells (tissues) in which they were synthesized. Thus, understanding the control of plant hormone synthesis is important in determining the relationship between synthesis and action.

Gibberellins (GAs) were originally discovered as phytotoxins in 1920s by Japanese phytopathologists. The pathogenic fungus, Gibberella fujikuroi, infects rice plants and secretes a compound that causes pathological longitudinal growth (Bakanae “mad seedling disease”). Between 1935 and 1938 the active substance was isolated and crystallized. It was called “gibberellin.” Later researches showed that GAs are also synthesized by higher plants and are very important in the regulation of growth and in differentiation processes.

The basic structure of approximately 80 GAs identified up to 1992 is the tetracyclic ring system of the ent-gibberellan (FIG. 1 a). GAs contain diterpenoid carboxylic acids produced mainly from mevalonic acid via cyclization of geranylgeranyl pyrophosphate (FIG. 1 b). Most of the GAs are inactive in promoting plant development. In many plants, biologically active GAs, which act as plant growth regulators, are GA₁ and GA₄. They can control various developmental processes, including seed germination, stem elongation, flowering, and fruit development. Thus, various modified plants that are industrially useful can be generated by modifying GA biosynthesis.

The role of GAs as mediators of environmental stimuli has been well established. Physical factors, such as light and temperature, can modify GA metabolism by changing the flux through specific step in the pathway. For example, light quality (red or far-red) and intensity (high or low) affects GA biosynthesis. In lettuce seeds and cowpea epicotyls, 3-beta hydroxylation of GA₂₀ is enhanced by treatment with far-red light (Toyumasu et al., (1992) Plant Cell Physiol. 33, 695–701). In addition, when pea seedlings are grown in low irradiance (40 μmol/m²s), GA₂₀ content increases sevenfold compared with plants grown in high irradiance (386 μmol/m²s), whereas in plants grown in the dark, the GA₂₀ content is reduced as compared to that in high irradiance (Gawronska et al., (1995) Plant Cell Physiol. 36, 1361–1367).

In spite of many attempts to implicate GA metabolism in phytochrome-mediated or light intensity-mediated changes in growth rate, supporting evidence is sparse. The mechanism(s) underlying these regulatory processes will eventually be understood as a result of the current advances in the molecular biology of GA biosynthesis.

Despite considerable efforts, the site of synthesis of bioactive GAs and their mode of action in specific cells and tissues has not been clarified. Based on experiments in which plants were supplemented with ¹⁴C-labelled GAs, it is thought that they are translocated in a non-polar manner throughout the plant. More recently, grafting experiments with dwarf and wild-type pea plants indicated that GA₁, one of the bioactive GA, is not transported, unlike its precursor, GA₂₀, (Proebsting et al. (1992) Plant Physiol. 100, 1354–1360; Reid et al., (1983) J. Exp. Bot. 34, 349–364). Quantitative analyses using GC-MS and bioassays with dwarf pea plants have revealed that GAs are mainly present in actively growing and elongating tissues such as shoot apices, young leaves, and flowers (Jones and Phillips, (1966) Plant Physiol. 41, 1381–1386; Potts et al. (1982) Physiol. Plant, 55, 323–328: Kobayashi et al., (1988) Agric. Biol. Chem. 52, 1189–1194). However the exact amount of each GA in a specific tissue is difficult to determine because most GAs are present in very small amounts and most are not bioactive. Therefore, a new approach is needed to clarify the location of synthesis of bioactive GAs.

According to progress in molecular biology and genetic engineering, almost of the genes encoding GA biosynthetic enzymes so far have been cloned from various plant species. The studies of these clones have shown that GA responsive dwarf mutants lack the respective GA biosynthetic enzymes (FIG. 1 b). Their expression profile indicates that the pathway is strictly regulated during development. Among these genes, GA1 from Arabidopsis, which encodes copalyl diphosphate synthase (CPS), an enzyme active early in GA biosynthesis, is highly expressed in rapidly growing tissues, e.g., the shoot apex, root tips, and flowers (Silverstone et al., (1997) Plant J. 12, 9–19). GA C-20 oxidase, which catalyzes a late step in the GA biosynthetic pathway and constitutes a small gene family, is specifically expressed in stems and developing seeds of Arabidopsis, pea, and bean where GA is required for development. They are negatively regulated by treatment with GA₃ (Phillips et al., (1995) Plant Physiol. 108, 1049–1057; Garcia-Martinez et al., (1997) Plant Mol. Biol. 33, 1073–1084).

These observations lead the present inventors to speculate that GA action in various organs may depend upon the amount of endogenous GA present, which in turn depends on the regulation of expression of GA biosynthetic enzymes, rather than on the translocation of bioactive GA to the site of GA action. However, analysis of the expression of CPS or GA C-20 oxidase provides no direct evidence of the site of synthesis of bioactive GAs and regulation of bioactive GA levels because bioactive GAs are synthesized by 3β-hydroxylation which is catalyzed by GA 3β-hydroxylase.

As described above, 3β-hydroxylase catalyzes the conversion of the GA₂₀ and GA₉ to GA₁ and GA₄, respectively, at the final step in the synthesis of bioactive GAs (FIG. 1 b). The enzymology of 3β-hydroxylase has not yet been completely clarified. However, the 2-oxoglutarate-binding region is essential for its activity, indicating that GA 3β-hydroxylase has the typical properties of a 2-oxoglutarate-dependent dioxygenase. Certain GA 3β-hydroxylase may be multifunctional; the enzyme from pumpkin endosperm catalyzes both 2β and 3β hydroxylation (Lange et al., (1997) Plant Cell, 9, 1459–1467). Maize dwarf-1,3β-hydroxylase has also been considered to be multifunctional and it catalyzes three hydroxylation steps in the maize GA biosynthetic pathway (Spray et al., (1996) Proc. Natl. Acad. Sci. 93, 10515–10518). But the nature of these GA 3β-hydroxylases is still not well known.

DISCLOSURE OF THE INVENTION

The present invention provides novel GA 3β-hydroxylase genes from rice and uses of the genes, especially for production of plants whose plant type has been modified.

For the isolation of GA 3β-hydroxylase genes from rice, the present inventors first performed PCR using degenerate primers designed based on the conserved region of dicot GA 3β-hydroxylases with the rice genomic DNA as a template. Then, using a fragment of the genomic DNA encoding GA 3β-hydroxylase thus obtained as a probe, the present inventors screened the rice genomic library to obtain several clones. These clones were divided into two groups based on their restriction maps, and one clone of each group was entirely sequenced. As a result, the present inventors found out that each of these clones encodes one rice GA 3β-hydroxylase.

Then, to obtain cDNA fragments based on the nucleotide sequence of each clone, the present inventors performed RT-PCR using the total RNA isolated from the rice seedlings (rice shoot apices) or unopened flowers, thereby obtaining the full-length cDNA clones encoding GA 3β-hydroxylases (designated “Os3β-1” and “Os3β-2”, respectively). Furthermore, using primers designed based on the genomic DNA sequences thus obtained, the present inventors performed RT-PCR with the total RNA isolated from the rice seedlings (shoot apices) or unopened flowers as a template and succeeded in obtaining the cDNA clones encoding the complete rice GA 3β-hydroxylase.

Since in rice the d18 mutant is known as a GA-responsive dwarf cultivar, the present inventors examined whether the rice GA 3β-hydroxylase clone thus isolated corresponds to the D18 gene. RFLP (Restriction Fragment Length Polymorphism) analysis and direct analysis of the nucleotide sequence of d18 allele proved that, of the two isolated rice genes, the Os3β-2 gene is the causative gene of the d18 mutation. In addition, the difference between the Os3β-1 gene and the Os3β-2 gene in their expression plant parts indicated that the Os3β-1 protein is involved in a different biosynthetic pathway of bioactive GAs than the Os3β-2 protein.

Furthermore, the present inventors succeeded in producing a dwarfed rice plant compared with a wild type plant by suppressing the expression of the Os3β-2 gene in rice plant utilizing the antisense DNA of the gene.

As described above, the present inventors succeeded in isolating a novel GA 3β-hydroxylase gene from rice, and found that a plant with modified plant type compared with a wild type plant can be produced by suppressing the expression of the gene.

In more detail, the present invention is to provide:

(1) a DNA encoding a protein having the gibberellin 3β-hydroxylase activity according to any one of the following (a) through (c):

-   -   (a) a DNA encoding a protein comprising the amino acid sequence         set forth in SEQ ID NO: 1 or 2,     -   (b) a DNA comprising the coding region of the nucleotide         sequence set forth in SEQ ID NO: 3 or 4.     -   (c) a DNA encoding a protein comprising the amino acid sequence         set forth in SEQ ID NO: 1 or 2 in which one or more amino acids         are substituted, deleted, added, and/or inserted;

(2) a DNA encoding an antisense RNA complementary to the DNA according to (1) or its transcription product;

(3) a DNA encoding an RNA having the ribozyme activity to specifically cleave the transcription product of the DNA according to (1);

(4) a DNA encoding an RNA that suppresses the expression of endogenous DNA according to (1) by co-suppression when the endogenous DNA is expressed in plant cells;

(5) a vector containing the DNA according to any one of (1) through (4);

(6) a transformed plant cell harboring the DNA according to any one of (1) through (4) in an expressible state;

(7) a transgenic plant containing the transformed plant cell according to (6);

(8) a propagative material of the transgenic plant according to (7);

(9) a protein encoded by the DNA according to (1);

(10) a method for producing the protein according to (9), wherein said method comprises culturing the transformed cells carrying the DNA according to (1) in an expressible state, and recovering the expressed protein from said cells or the culture supernatant thereof;

(11) a method for modifying the plant growth, wherein said method comprises controlling the expression level of the DNA according to (1) in plant cells; and

(12) a method for modifying a plant type, wherein said method comprises controlling the expression level of the DNA according to (1) in plant cells.

The present invention provides novel GA 3β-hydroxylases isolated from rice plant and DNAs encoding these enzymes. Nucleotide sequences of cDNAs GA 3β-hydroxylase genes from rice, Os3β-1 and Os3β-2, isolated by the present inventors and included in the DNA of this invention, are set forth in SEQ ID NOs: 3 and 4, respectively. The nucleotide sequence of the genomic DNAs of these cDNAs are shown in SEQ ID NOs: 5 and 6, respectively. In addition, amino acid sequences of the “Os3β-1” and “Os3β-2” proteins are set forth in SEQ ID NOs: 1 and 2, respectively.

Consistent with the previous classification of the 3β-hydroxylase as 2-oxoglutarate-dependent dioxygenases (2-ODDs), both “Os3β-1” and “Os3β-2” proteins originating in rice contained all of the domains characteristic of plant 2-ODDs (Prescott, A. G. (1993) J. Exp. Bot. 44, 849–861; de Carolis and Luca (1994) Phytochemistry 36, 1093–1107). Amongst all of the published sequences, coding regions of both clones show the highest homology with GA 3β-hydroxylases. Especially, these regions are highly conserved (position of 240 to 247 and 302 to 307 in Os3β-1, 222 to 229 and 285 to 290 in Os3β-2) and may act as the binding site of iron and the cofactor, 2-oxoglutarate. These proteins also have the conserved motif (Met-Trp-X-Glu-Gly-X-Thr), which is unique to the GA 3β-hydroxylase (position of 144 to 150 in Os3β-1, 127 to 133 in Os3β-2). Sequence comparison with dicot GA 3β-hydroxylases and other dioxygenases suggests that both cDNA clones isolated by the present inventors encode rice GA 3β-hydroxylases.

Mapping of Os3β-2 and genomic Southern analysis indicated that Os3β-2 corresponds to the D18 gene. The rice d18 mutants are GA-responsive dwarf cultivars, including Hosetu-waisei, Akibare-waisei, Kotake-tamanishiki, and Waito-C (FIG. 2). Many dwarf alleles so far have been identified. The Os3β-2 protein is assumed to be also involved in the internodal growth of plants via synthesis of bioactive GAs.

Analyses on the levels of endogenous GAs in different organs at various growth stages have revealed that 13-hydroxylated gibberellins (GA₁₉, GA₂₀, GA₁) are dominant in vegetative organs, while non-13-hydroxylated gibberellins (GA₂₄, GA₉, GA₄) are specifically accumulated in reproductive growth organs, especially in anthers. This indicates that the biosynthetic pathway for bioactive GAs is organ-specific (Kurogouchi, S. et al. (1979) Planta 146, 185–191; Kobayashi, M. et al. (1984) Agric. Biol. Chem. 48, 2725–2729; Kobayashi, M. et al, (1988) Agric. Biol. Chem. 52, 1189–1194). Expression patterns of Os3β-2 (D18) and Os3β-1 are consistent with this speculation. Indeed, the Os3β-2 mRNA was in high level in stems, young leaves, and inflorescence meristems, while the Os3β-1 mRNA was specifically observed in flowers. These consistencies indicate that products of Os3β-2 and Os3β-1 may have the substrate-specificity for GA₂₀ and GA₉, respectively.

Expressions of Os3β-2 and Os3β-1 are also consistent with the distribution of bioactive GAs in rice. Breeding and quantitative analyses revealed that GA₁ was high in young leaf tissues which are the most active sites of gibberellin biosynthesis (Choi, Y.-H. et al. (1995) Plant Cell Physiol. 36(6), 997–1001), and the highest expression of Os3β-2 was also observed in young leaves. Interestingly, the expression of Os3β-2 in shoot apices was at moderate levels as compared with other organs of rice, nevertheless many genes for GA biosynthesis, such as GA1 from Arabidopsis and Nty from tobacco, are strongly expressed in the actively dividing and elongating tissues, like shoot apices and roots (Silverstone et al. (1997) Plant J. 12, 9–19). This discrepancy suggests that the organ activity to synthesize GAs may differ between monocotyledonous and dicotyledonous plants. On the other hand, the endogenous level of GA₄ is extremely high in anthers at the flowering stage (Kobayashi, M. et al. (1988) Agric. Biol. Chem. 52, 1189–1194; Kobayashi, M. et al. (1990) Plant Cell Physiol. 31(2), 289–293). This fact is consistent with the specific expression of Os3β-1 in flowers. This consistency may indicate that GA₄ is synthesized in anthers by the action of the Os3β-1 protein. Therefore, the Os3β-2 and Os3β-1 proteins may be involved in different biosynthetic pathways of bioactive GAs.

In fact, the Os3β-1 protein catalyzed the production of GA₄ (3β-hydroxylation), GA₇ (2,3-unsaturation and 3β-hydroxylation), and GA₃₄ (2β-hydroxylation) with GA₉ as its substrate (FIG. 9), and similar results were obtained with GA₂₀ as the substrate. In addition, with GA₅ and GA₄₄ as its substrate, the protein produced the corresponding 3β-hydroxylated gibberellins, GA₃ and GA₈₈, respectively. Furthermore, the Os3β-2 protein catalyzed, with GA₅, GA₉, GA₂₀, and GA₄₄ as the substrates, the production of the corresponding 3β-hydroxylated gibberellins, GA₃, GA₄, GA₁, and GA₃₈, respectively (FIG. 10) (Example 5).

Several reports mention the necessity of bioactive GAs not only for the stem elongation but also for a variety of other plant growth processes. For example, as to the floral organs, the male-sterile phenotype shown in an Arabidopsis GA-deficient mutant, gal-3 (Koornneef, M. and Van der Veen, J. H. (1980) Theor. Appl. Genet. 58, 257–263), and the arrest of anther growth at an early stage without formation of viable pollen grains in tomato mutants, stamenless-2 and gib-1 (Sawhney (1974) J. Exp. Bot. 25, 1004–1009; Jacobsen and Olszewski (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9292–9296), have been reported.

Since the proteins of the present invention are thought to be involved in the biosynthesis of bioactive GAs, they can be used in manufacturing bioactive GAs. Furthermore, as described below, the plant growth can be modified by regulating expression levels of these proteins in plants. For example, a plant having a different plant type from a wild type can be produced.

The protein of this invention can be prepared as a recombinant protein via methods known to those skilled in the art utilizing the gene recombination techniques or as a natural protein. A recombinant protein can be prepared, as described below, for example, by inserting DNA (e.g., SEQ ID NOs: 3 and 4) encoding the protein of this invention into an appropriate expression vector and purifying the protein from cells transformed with the vector. A natural protein can be prepared, for example, by immunizing suitable animals with the prepared recombinant protein or its partial peptide, binding the thus prepared antibody to a column for affinity chromatography, contacting the column with extracts prepared from tissues of tobacco and rice expressing the protein of this invention, and purifying the protein binding to the column.

The protein of this invention includes wild type proteins (SEQ ID NOs: 1 and 2) in which partial amino acid residues are modified, while retaining the function of the wild type proteins. An example of the method for preparing such modified proteins well known to those skilled in the art include the site-directed mutagenesis method (Kramer, W. & Fritz, H.-J. Oligonucleotide-directed construction of mutagenesis via gapped duplex DNA. Methods in Enzymology, 154: 350–367, 1987). Amino acid mutations may also occur spontaneously. The protein of this invention thus include proteins that retain the GA 3β-hydroxylase activity of the wild-type protein and those that are modified via substitution, deletion, addition, and/or insertion of one or more amino acid residues in the amino acid sequence of the wild type protein. There is no particular limitation on the site and number of such amino acid modifications in the protein so far as the modified protein retains the GA 3β-hydroxylase activity. The number of amino acid that can be modified is usually not more than 50 amino acid residues, preferably not more than 30, more preferably not more than 10, and most preferably not more than 3 amino acid residues.

Herein, the term “GA 3β-hydroxylase activity” refers to the activity to synthesize GA₁ or GA₄ as the reaction product when GA₂₀ or GA₉ is used as a reaction substrate and ferrous iron and 2-oxoglutarate are used as cofactors. The activity can be detected, for example, as follows. In general, cDNA obtained is inserted into an expression vector and overexpressed as a fusion protein in E. coli. Using the cell extract thus obtained (as an enzyme solution), the reaction is performed in vitro with GA₂₀ or G₉ as a reaction substrate in the presence of the co-factors, ferrous ion and 2-oxoglutarate, and finally the reaction product (GA₁ or GA₄) is confirmed by GC-MS.

The present inventors isolated cDNA and genomic DNA encoding the above proteins. Therefore, DNAs encoding the protein of this invention include both cDNA and genomic DNA so far as they encode these proteins. When DNAs encoding the Os3β-2 and Os3β-1 proteins are cDNAs, the DNAs can be prepared by RT-PCR using respective primers designed based on the information of nucleotide sequences set forth in SEQ ID NOs: 3 and 4 and the total RNA isolated from seedlings (shoot apices of rice) or unopened flowers as a template. Furthermore, the genomic DNA can be prepared by PCR using respective primers designed based on the information of nucleotide sequences set forth in SEQ ID NOs: 5 and 6 and the rice genomic DNA as a template.

DNAs encoding the protein of the present invention can be used, for example, for producing recombinant proteins. Production of recombinant proteins can be carried out described below. First, a full-length cDNA is synthesized by RT-PCR using primers provided with restriction enzyme sites and subcloned into multi-cloning sites of the pMAL-c2 expression vector (NEB). This construct is used to transform Escherichia coli strain BL21 cells (protease-deficient strain) by standard methods. Using the transformant thus obtained, the protein is induced. E. coli are cultured (by shaking) in a 2×YT medium containing 0.2% glucose at 37° C. When an OD₆₀₀ value reaches around 0.6, IPTG is added to a final concentration of 1 mM, and culturing is further continued at 18° C. for 24 h. Extraction of an enzyme solution is performed as follows. After culturing, cells are collected and lysed in a suspension buffer (50 mM Tris-HCl (pH 8.0) containing 10% glycerol, 2 mM DTT, and 1 mg/ml lysozyme). The cell suspension is allowed to stand at 4° C. for 30 min, and then incubated at −80° C. until it becomes completely frozen. The frozen suspension is thawed and sonicated for 30 s twice at 5-min intervals at the MAX level with the Sonicator (Heat Systems—Ultrasonics, Inc., Model W-225R). The suspension thus treated is centrifuged (at 15,000 rpm and 4° C. for 20 min), and the supernatant is used as a crude enzyme solution.

Furthermore, preparation of the purified protein can be carried out, by expressing the protein of this invention in E. coli (or the like) as a fusion protein with the histidine tag, maltose-binding protein, or glutathione-S-transferase (GST), and subsequently purifying them on a nickel column, an amylose-column, or a GST-glutathione column, respectively. Then, after the purification, the above-described tags can be cleaved off using limited proteases, such as, thrombin and factor Xa as required.

As described above, the genes isolated by the present inventors are assumed to be involved in the plant growth through the production of bioactive GAs. Therefore, plant growth may be controlled by regulating the expression of these genes. Since Os3β-2 in particular is thought to be involved in the internodal growth of plants, this gene may be utilized in the control of plant stature. Control of plant stature provides a variety of industrial advantages.

For example, the shortened stature caused by suppressing the expression of the gene of this invention in a plant can make the plant resistant to bending thereby increasing the fruit weight. Furthermore, the shortened stature makes the size of the plant per stub more compact so that the number of plants to be planted per unit area can be increased. This dense planting is highly important in the production of agricultural products including rice, wheat, maize, etc., in particular. DNA encoding the protein of the present invention may be applicable to dwarf flowering plants, dwarf fruit trees, etc. Male sterile traits may be induced by suppressing the expression of the Os3β-1 gene in flowers.

On the other hand, the yield of plants as a whole may be enhanced by lengthening plant stature through the elevated expression of genes of this invention within the plants. This is useful for improving feed crop yields as a whole in particular.

In the present invention, a variety of methods known to those skilled in the art are available for suppressing the expression of genes of this invention to control plant growth. Herein, “suppression of expression of genes” includes suppressions of both gene transcription and translation into proteins, and includes not only complete suppression but also decrease in the gene expression.

The expression of a specific endogenous gene in plants can be suppressed by conventional methods utilizing antisense technology. Ecker et al. were the first to demonstrate the effect of an antisense RNA introduced by electroporation in plant cells by using the transient gene expression method (Ecker, J. R. and Davis, R. W. (1986). Proc. Natl. Acad. Sci. USA 83, 5372). Thereafter, the target gene expression was reportedly reduced in tobacco and petunias by expressing antisense RNAs (van der Krol, A. R. et al. (1988). Nature 333, 866). The antisense technique has now been established as a means to suppress target gene expression in plants.

Multiple factors cause antisense nucleic acid to suppress the target gene expression. These include inhibition of transcription initiation by triple strand formation; suppression of transcription by hybrid formation at the site where the RNA polymerase has formed a local open loop structure; transcription inhibition by hybridization with the RNA being synthesized; suppression of splicing by hybrid formation at the junction between an intron and an exon; suppression of splicing by hybrid formation at the site of spliceosome formation; suppression of mRNA translocation from the nucleus to the cytoplasm by hybridization with mRNA; suppression of splicing by hybrid formation at the capping site or at the poly A addition site; suppression of translation initiation by hybrid formation at the binding site for the translation initiation factors; suppression of translation by hybrid formation at the site for ribosome binding near the initiation codon; inhibition of peptide chain elongation by hybrid formation in the translated region or at the polysome binding sites of mRNA; and suppression of gene expression by hybrid formation at the sites of interaction between nucleic acids and proteins. These factors suppress the target gene expression by inhibiting the process of transcription, splicing, or translation (Hirashima and Inoue, “Shin Seikagaku Jikken Koza (New Biochemistry Experimentation Lectures) 2, Kakusan (Nucleic Acids) IV, Idenshi No Fukusei To Hatsugen (Replication and Expression of Genes),” Nihon Seikagakukai Hen (The Japanese Biochemical Society Ed.), Tokyo Kagaku Dozin, pp. 319–347, (1993)).

An antisense sequence used in the present invention can suppress the target gene expression by any of the above-mentioned mechanisms. If an antisense sequence is designed to be complementary to the untranslated region near the 5′ end of the gene's mRNA; it will effectively inhibit translation of a gene. Additionally, it is also possible to use sequences that are complementary to the coding regions or to the untranslated regions on the 3′ side. Thus, the antisense DNA used in the present invention includes a DNA having antisense sequences against both the untranslated regions and the translated regions of the gene. The antisense DNA to be used is connected downstream from an appropriate promoter, and, preferably, a sequence containing the transcription termination signal is connected on the 3′ side. The DNA thus prepared can be transfected into the desired plant by known methods. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene (or the homologue) of the plant to be transformed or a part thereof, but it need not be perfectly complementary so long as it can effectively inhibit the gene expression. The transcribed RNA is preferably not less than 90%, and most preferably not less than 95% complementary to the transcribed products of the target gene. In order to effectively inhibit the expression of the target gene by means of an antisense sequence, the antisense DNA should be at least 15 nucleotides long or more, preferably 100 nucleotides long or more, and most preferably 500 nucleotides long or more. The antisense DNA to be used is generally shorter than 5 kb, and preferably shorter than 2.5 kb.

DNA encoding ribozymes can also be used to suppress the expression of endogenous genes. A ribozyme is defined as an RNA molecule that has catalytic activities. Numerous ribozymes are known in the literature, each having distinct catalytic activity. Research on the ribozymes as RNA-cleaving enzymes has enabled the designing of a ribozyme that site-specifically cleaves RNA. While some ribozymes of the group I intron type or the M1RNA contained in RNaseP consist of 400 nucleotides or more, others belonging to the hammerhead type or the hairpin type have an activity domain of about 40 nucleotides (Koizumi, Makoto and Ohtsuka, Eiko (1990). Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme) 35, 2191).

The self-cleavage domain of a hammerhead type ribozyme cleaves at the 3′ side of C15 sequence G13U14C15. Formation of a nucleotide pair between U14 and A at the ninth position is considered important for the ribozyme activity. Furthermore, it has been shown that the cleavage also occurs when the nucleotide at the 15th position is A or U instead of C (Koizumi, M. et al. (1988). FEBS Lett. 228, 225). If the substrate-binding site of the ribozyme is designed to be complementary to the RNA sequences adjacent to the target site, one can create a restriction-enzyme-like RNA cleaving ribozyme that recognizes the sequence UC, UU, or UA within the target RNA (Koizumi, M. et al. (1988). FEBS Lett. 239, 285; Koizumi, Makoto and Ohtsuka, Eiko (1990). Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme), 35, 2191; Koizumi, M. et al. (1989). Nucleic Acids Res. 17, 7059). For example, in the coding region of the Nty gene, Os3β-1 gene, or Os3β-2 gene (SEQ ID NO: 3 or 4) isolated by the present inventors, there are pluralities of sites that can be used as the ribozyme target.

The hairpin type ribozyme is also useful in the present invention. A hairpin type ribozyme can be found, for example, in the minus strand of the satellite RNA of tobacco ringspot virus (Buzayan, J. M. (1986). Nature 323, 349). This ribozyme has also been shown to target-specifically cleave RNA (Kikuchi, Y. and Sasaki, N. (1992). Nucleic Acids Res. 19, 6751; Kikuchi, Yo (1992) Kagaku To Seibutsu (Chemistry and Biology) 30, 112).

The ribozyme designed to cleave the target is fused with a promoter, such as the cauliflower mosaic virus 35S promoter, and with a transcription termination sequence, so that it will be transcribed in plant cells. However, if extra sequences are added to the 5′ end or the 3′ end of the transcribed RNA, the ribozyme activity may be lost. In this case, one can place an additional trimming ribozyme, which functions in the cis position to perform the trimming on the 5′ or the 3′ side of the ribozyme portion, thereby precisely cutting the ribozyme portion from the transcribed RNA containing the ribozyme (Tairas, K. et al. (1990). Protein Eng. 3, 733; Dzaianott, A. M. and Bujarski, J. J. (1989). Proc. Natl. Acad. Sci. USA 86, 4823; Grosshands, C. A. and Cech, R. T. (1991). Nucleic Acids Res. 19, 3875; Taira, K. et al. (1991.) Nucleic Acid Res. 19, 5125). Multiple sites within the target gene can be cleaved by arranging these structural units in tandem to achieve greater effects (Yuyama, N. et al., (1992). Biochem. Biophys. Res. Commun. 186, 1271). By using such ribozymes, it is possible to specifically cleave the transcription products of the target gene in the present invention, thereby suppressing the expression of the gene.

Endogenous gene expression can also be suppressed by co-suppression through the transformation by DNA having a sequence identical or similar to the target gene sequence. “Co-suppression,” as used herein, refers to the phenomenon in which, when a gene having a sequence identical or similar to the target endogenous gene sequence is introduced into plants by transformation, expression of both the introduced exogenous gene and the target endogenous gene becomes suppressed. Although the detailed mechanism of co-suppression is unknown, it is frequently observed in plants (Curr. Biol. (1997). 7, R793, Curr. Biol. (1996). 6, 810). For example, if one wishes to obtain a plant body in which the gene of the present invention is co-suppressed, the plant in question can be transformed with a DNA vector designed so as to express the gene of the present invention or DNA having a similar sequence. The gene to be used for co-suppression need not be completely identical to the target gene. However, it should have preferably 70% or more sequence identity, more preferably 80% or more sequence identity, and most preferably 90% or more (e.g. 95% or more) sequence identity.

The identity of one amino acid sequence or nucleotide sequence to another can be determined by following the BLAST algorithm by Karlin and Altschl (Proc. Natl. Acad. Sci. USA, (1993). 90, 5873–5877,). Programs such as BLASTN and BLASTX were developed based on this algorithm (Altschul et al. (1990). J. Mol. Biol.215, 403–410). To analyze a nucleotide sequences according to BLASTN based on BLAST, the parameters are set, for example, as score=100 and word length=12. On the other hand, parameters used for the analysis of amino acid sequences by the BLASTX based on BLAST include, for example, score=50 and word length=3. Default parameters of each program are used when using BLAST and Gapped BLAST programs. Specific techniques for such analysis are known in the art [available on the website of the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Bethesda Md. 20894, U.S.A.].

Modification of plant growth utilizing a DNA functioning to suppress the expression of genes of this invention may be achieved by inserting the DNA into an appropriate vector, transferring the vector into plant cells, and regenerating the transformed plant cells thus obtained. There is no particular limitation on the type of vectors so far as they are capable of expressing the inserted gene within plant cells.

For example, the promoter for the gene isolated by the present inventors may be used. A vector having a promoter (for example, 35S promoter of cauliflower mosaic virus) that enables the constitutive gene expression in plant cells may also be used. Furthermore, plant tissue-specific promoters may specifically modify particular plant tissues, for example, leaves, flowers, fruits, etc. Examples of the tissue-specific promoters are seed-specific promoters such as promoters for β-phaseolin of kidney bean (Bustos, et al. (1991). EMBO J. 10, 1469–1479) and glycinin of soy bean (Lelievre, et al. (1992). Plant Physiol. 98, 387–391); leaf-specific promoters such as promoters for the RbcS gene of pea (Lam and Chua (1990). Science 248, 471–474) and Cab 1 gene of wheat (Gotorn, et al. (1993). Plant J. 3, 509–518), root-specific promoters such as promoters for the TobRB7 gene of tobacco (Yamamoto, et al. (1991). Plant Cell 3, 371–382) and rolD gene of Agrobacterium rhizogenes (Elmayan and Tepfer (1995). Transgenic Res. 4, 388–396). It is also possible to use a vector having a promoter inducibly activated by exogenous stimuli.

Although there is no particular limitation on the type of plant cells into which a vector is inserted, rice and tobacco, from which the genes of the present invention are derived, are particularly preferred. Herein, the term “plant cells” includes plant cells in a variety of forms, for example, cultured cell suspension, protoplasts, leaf sections, cali, etc. A vector can be transferred into plant cells by a variety of methods well known to those skilled in the art, including the polyethylene glycol method, electroporation method, Agrobacterium-mediated method, particle gun method, etc. Regeneration of a plant body from transformed plant cells may be performed by the standard methods known in the art. Once the transformed plant body is generated, it is possible to obtain propagative materials (for example, seeds, tubers, cuttings, etc.) from the plant body and produce the transformed plant of this invention on a large scale.

Furthermore, in this invention, it may also be possible to enhance the plant growth by promoting the expression of DNA isolated by the present inventors. In this case, the DNA is inserted into an appropriate vector, and the resulting recombinant vector is transferred into plant cells so as to regenerate the transformed plant cells thus obtained. Vectors used for the expression in plant cells, plant cells into which vectors are transferred, methods for regenerating plant bodies are similar to those in the case where the above-described antisense DNAs and the like are used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows a general structure of gibberellin (ent-gibberellan).

FIG. 1 b shows a major GA biosynthetic pathway in higher plants. The italicized letters indicate a GA-responsive dwarf mutant that lacks a specific GA biosynthetic pathway: ga1, ga2, ga3, ga5, and ga4 from Arabidopsis; an1, d5, d3, and d1 from maize; ls and le from pea; d35 (dx) and d18 (dy) from rice.

FIG. 2 is a photograph showing various phenotypes of d18 dwarf plants. From left to right, Taichuu-65 (WT: approximately 1 m at the final stage), Kotake-tamanishiki dwarf (d18^(k): approximately 65 cm), Waito-C (approximately 55 cm), Hosetsu-waisei dwart (d18^(h): approximately 15 cm), and Akibare-waisei dwart (Bar=10 cm).

FIG. 3 shows the loci of GA 3β-hydroxylase (Os3β-1 and Os3β-2) genes and various RFLP makers on rice chromosomes 1 and 5.

FIG. 4 a is an electrophoretogram representing the results of RFLP analysis of the D18 and d18 alleles. DNA was isolated from leaf tissues of D18 (Shiokari and Akibare) and d18 allele (d18^(h), ld18^(h), and d18-AD) plants. The DNA was digested with ApaI, separated by electrophoresis, bound to nylon filters, and then hybridized. Molecular length markers are given at left in kilobases.

FIG. 4 b is a restriction map of genomic Os3β-2 clone and its subclone. Genomic clone was obtained by screening the genomic library using the PCR product as a probe. The 2.3-kb BglII fragment subclone contains the entire coding region of D18.

FIG. 5 is an electrophoretogram representing expression patterns of GA 3β-hydroxylases in wild type Oryza sativa plants. The patterns were obtained by hybridization of 3β-hydroxylase cDNA D18 and Os3β-1 to northern blots from 10 μg of total RNA extracted from SA (shoot apices), ST (stems), LB (leaf blades), Ra (rachises), FL (flowers), YL (young leaves), IFM (inflorescence meristems), and Sh (2-week-old seedlings).

FIG. 6 is a photograph representing transformed plants in which the Os3β-2 (D18) cDNA is constitutively expressed in the antisense orientation under the control of the actin promoter. A wild type nipponbare plant is on the left, a semi-dwarfed plant on the middle, and a dwarfed plant on the right.

FIG. 7 shows the plasmid pBS-SK⁺ into which the full-length Os3β-2 cDNA has been inserted.

FIG. 8 shows the plasmid pAct-NOS/Hm2 into which the antisense Os3β-2 (D18) gene has been inserted.

FIG. 9 shows the pathway of the production of GA₄, GA₇, and GA₃₄ catalyzed by an Os3β-1 fusion protein when GA₉ is used as a substrate.

FIG. 10 shows the pathway of the production of 3β-hydroxylated gibberellins (GA₃, GA₄, GA₁, and GA₃₈) corresponding to GA₅, GA₉, GA₂₀, and GA₄₄, respectively, which are used as substrates, catalyzed by an Os3β-2 fusion protein.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail below with reference to Examples, but is not to be construed as being limited thereto. Rice seeds (Oryza sativa, Japonica-type cultivars: “Nipponbare”, “Akibare”, “Shiokari” and others) were sterilized in 1% NaClO for 1 h and thoroughly rinsed in sterile distilled water and germinated on soil and grown at a greenhouse.

EXAMPLE 1 Isolation of cDNA Clone Encoding GA 3β-hydroxylase

No report on isolation of GA 3β-hydroxylase in monocots is available. Several GA 3β-hydroxylases have been cloned from dicots (Chiang et al., 1995; Martin et al., 1997; Lester et al., 1997).

For the isolation of a partial fragment encoding GA 3β-hydroxylase, PCR was performed using the rice genomic DNA as a template and degenerate primers (5′ primer: 5′-GTNGTNAARGTNGGNGARRT-3′/SEQ ID NO: 7; 3′ primer: 5′-AYYTARTCRTTGGANGTNAC-3′/SEQ ID NO: 8) designed from the conserved region among the reported dicot's GA 3β-hydroxylase sequences. A 210-bp DNA fragment, which corresponds to the size expected from the reported GA 3β-hydroxylase sequences, was obtained.

To isolate full-length clones, the present inventors screened the rice genomic library with this fragment as a probe. Several clones were isolated and were divided into two groups based on the restriction map of each genomic clone. Finally one clone of each group was entirely sequenced and designated as Os3β-1 and Os3β-2 (Os3β-1 and Os3β-2; Oriza sativa GA 3β-hydroxylase -1, -2). Nucleotide sequences of these clones were set forth in SEQ ID NOs: 5 and 6. Os3β-1 shared the sequence with the fragment used as a probe, but Os3β-2 contained a different sequence at the corresponding region from the fragment.

Based on the sequence of each clone, the present inventors performed RT-PCR using the total RNA isolated from seedlings (rice shoot apices) or unopened flowers to obtain cDNA fragment. As a result, full-length cDNA clones encoding GA 3β-hydroxylases were obtained. Each cDNA, Os3β-1 or Os3β-2, contained an open reading frame encoding a polypeptide with 379 or 373 amino acids, respectively. Nucleotide sequences of these clones are set forth in SEQ ID NOs: 3 and 4. The Os3β-2 genomic DNA contained a single short intron (110 bp) and the intron was located at the same position as previously reported GA 3β-hydroxylases in dicot. The genomic DNA of the other clone, Os3β-1, contained two introns. One was located at the same position as the intron of Os3β-2 and its size was comparable to that of Os3β-2 (110 bp). The other was located at the position of the binding site of the co-factor, 2-oxoglutarate (400 bp) (data not shown). The deduced amino acid sequences of both clones shared a high degree of similarity to other GA 3β-hydroxylases, and they showed the highest similarity to each other (56.6% identity, and 88.2% similarity).

Nucleotide sequences were determined by the dideoxynucleotide chain-termination method using an automated sequencing system (ABI373A). Analysis of sequences was carried out using GENETYX computer software (Software Kaihatsu Co., Japan).

EXAMPLE 2 Identification and Characterization of d18 Alleles

Previous quantitative analyses and bioassay of the dwarf rice plants indicated that the D18 gene encodes a GA 3β-hydroxylase. The D18 locus has been identified on chromosome 1, flanking to the FS-2 locus at the bottom of this chromosome. To investigate that the isolated GA 3β-hydroxylase clones correspond to the D18 gene, the present inventors mapped two clones on the rice genome using RFLP (Restriction Fragment Length Polymorphism) analysis.

RFLP of Os3β-1 or Os3β-2 were present between Asominori (a Japonica rice) and IR 24 (an Indica rice) DNAs digested with EcoRI or ApaI, respectively. Linkage analysis was performed with digested genomic DNA from F2 progeny of crosses between Asominori and IR 24. Os3β-1 and Os3β-2 are mapped on the top of chromosome 5 and the bottom of chromosome 1, respectively (FIG. 3). This result suggests that the Os3β-2 locus corresponds to the D18 locus.

Further analysis was performed to confirm that Os3β-2 is located at the D18 locus. There are four independent conventional mutants generated by the loss-of-function of the D18 gene. The mutations may be caused by the DNA rearrangements and/or deletions of the D18 gene by mutagenesis using γ-irradiation. Therefore, RFLP at the position of Os3β-2 between a wild type and these mutants may be observed if Os3β-2 is the D18 gene.

DNA gel blot analysis of genomic DNA from a wild type (Shiokari or Akibare) and the d18 alleles (ld18^(k), ld18^(h), and d18-AD) was performed. ld18^(k) and ld18^(h) are isogenic lines of Shiokari background, and d18-AD was isolated from an Akibare mutant generated by mutagenesis with ethylenimines (EI). For DNA gel blot analysis, rice genomic DNA (1 μg per lane) was digested with restriction enzymes, separated by agarose gel electrophoresis, and transferred to Hybond N+ nylon membrane (Amersham) (Sambrook et al., 1989). Hybridization was performed at 65° C. in 0.25 M Na₂HPO₄, 1 mM EDTA, and 7% SDS. Filters were washed twice for 15 min at 65° C. in 2×SSC, 0.1% SDS and once in 0.1×SSC, 0.1% SDS at 65° C. for 15 min.

Eight enzymes (BamHI, BglII, ApaI, KpnI, DraI, EcoRV, EcoRI, and HindIII) were used to digest these genomic DNA to find RFLP between the wild type plants and the mutants. Polymorphisms were observed when the DNAs from d18-AD and ld18^(h) were digested with ApaI, while ld18^(k) did not show any polymorphisms when digested with any enzymes tested (FIG. 4).

The result of RFLP analysis strongly suggests that d18-AD has a long deletion in the D18 locus, while ld18^(h) has a short deletion including the ApaI site. To confirm the result, the present inventors analyzed the entire coding sequences of all d18 alleles and compared them with that of the wild type Os3β-2.

Specifically, oligonucleotide primers designed based on the 5′ and 3′ noncoding sequences of D18 were used to amplify the 1.6-kb fragments containing the entire coding region from D18, d18^(h), d18k, and d18-w, and then the amplified fragments were sequenced.

As expected, the Os3β-2 coding sequence was altered in all d18 alleles at various positions (Table 1), while the d18-AD produced no RCR product.

TABLE 1 Position in Nature of coding Consequence Allele mutation^(a) sequence of mutation d18-AD 7-kb absence of (Akibare- deletion a full length waisei) of D18 ORF d18^(h) GGG to GG, Gly²⁵¹ frameshift, (Hosetsu- 1-base addition of 38 waisei) deletion novel amino acids, truncated polypeptide d18^(k) CGC to TGC Arg¹⁴⁵ amino acid substitution, (Kotake- substitute Asp to Cys, tamanishiki) altered product d18-w 9-base deletion Val⁵⁷ to Arg⁵⁹ in-frame deletion, (Waito-C) absence of a 3-amino-acid residue segment altered product ^(a)Underlining denotes nucleotide substitution in the d18^(k) allele.

This supports the above result that d18-AD almost entirely lost the coding sequence (data not shown). In the sequence from ld18^(k), substitution of C to T at the nucleotide position 433 numbering from the start codon, converted Arginine-168 to Cystine. In d18-w, in-frame deletion of 9 nucleotides at the position of 169 to 177, resulted in deletion of 3 amino acids Valine-57 to Arginine-59. In ld18^(h), deletion of nucleotide G caused a reading frame shift. These results demonstrate that the Os3β-2 gene is located at the D18 locus, and encodes an GA 3β-hydroxylase.

EXAMPLE 3 Regulation of the Os3β-2 (D18) and Os3β-1 Genes During Plant Growth

To investigate the expression of the D18 and Os3β-1 genes during plant growth, RNA gel blot analysis was performed. For this analysis, total RNA was prepared from various organs or tissues by the standard method (Sambrook et al., 1989). RNA (10 μg per sample) was separated by gel electrophoresis and transferred to a Hybond N+ nylon membrane (Amersham). Hybridization was performed at 65° C. in a solution containing 5×SSC, 10% (w/v) dextran sulfate, 0.5% (w/v) SDS, 0.1 mg/ml denatured salmon sperm DNA. Filters were washed twice for 15 min at 65° C. in 2×SSC, 0.1% SDS and once in 0.1×SSC, 0.1% SDS at 65° C. for 15 min. A BssHII-PvuII (519 bp) fragment from full-length D18 cDNA and a KpnI-PvuII (310 bp) fragment from Osβ-1, were used as probes.

D18 gene was expressed in every organ tested (FIG. 5). The levels of expression were high in stems, young leaves, and inflorescence meristems, and low in leaf blades and rachises. In contrast, the Os3β-1 mRNA expression was specifically high in the flowers, and low in leaf blades and rachises.

Because D18 and Os3β-1 are highly similar to each other, the present inventors examined the degree of cross-hybridization by genomic southern analysis. When the respective specific probe was used to genomic southern hybridization, cross-hybridization was not detected (data not shown).

EXAMPLE 4 Production of Dwarfed Plant by Suppressing Expression of Os3β-2 (D18) Gene

A full-length Os3β-2 cDNA that had been cloned to the BamHI-HindIII site in the pBS-SK+ plasmid (FIG. 7), was cleaved out from the vector by the BamHI-HindIII digestion to collect the cDNA, and then its ends were blunted. This blunted full-length cDNA was inserted to the SmaI site of the pAct-NOS/Hm2 plasmid to construct a vector to express the antisense Os3β-2 gene (FIG. 8).

Agrobacterium strain EHA101 was transformed with this recombinant plasmid by the electroporation method. Germinating seeds of rice were contacted with the Agrobacterium strain and cultured in a selection medium containing kanamycin and hygromycin for 3 weeks to select resistant cells, which were transplanted to a re-differentiation medium so as to obtain dozens of transgenic plants. As a result, plants expressing the antisense Os3β-2 gene became dwarfed compared with the wild type rice plant (FIG. 6).

EXAMPLE 5 Function of Recombinant GA 3β-hydroxylase

cDNAs for the predicted protein-coding regions of the Os3β-1 and Os3β-2 genes were each inserted into the pMAL-c2 expression vector (New England Biolabs, Beverly, Mass.) in the sense orientation to obtain a fusion translation product. The constructs thus obtained, pMAL-Os3β-1 and pMAL-Os3β-2, were expressed in E. coli strain JM109. Bacterial cells were cultured by shaking in 2×YT medium containing 100 mg/L ampicillin at 37° C. overnight. Then, the culture was diluted 100-fold with a fresh 2×YT medium containing 100 mg/L ampicillin, and further cultured with shaking at 30° C. Four hours later, IPTG was added to the culture to a final concentration of 1 mM, and the mixture was incubated with shaking at 17° C. for further 18 h. After the completion of culturing, the bacterial cells were collected, washed with a washing buffer (containing 50 mM Tris-HCl (pH 8.0), 10% (w/v) glycerol, and 2 mM DTT), suspended in the washing buffer containing lysozyme (1 mg/ml), and allowed to stand for 30 min on ice.

The cell lysate thus obtained was sonicated, centrifuged, and the supernatant was subjected to SDS-PAGE to confirm the expression of the fusion protein. For the assay of enzymatic activity of the Os3β-1 fusion protein, the supernatant was incubated with various gibberellins and cofactors (ascorbate, ferrous iron, and 2-ketoglutarate). For the activity assay of the Os3β-2 fusion protein, the supernatant was purified on a column of amylose resin according to the method described in the manual, and the purified protein thus obtained was similarly incubated as described above. Metabolized gibberellins were identified using GC-MS. In the case of the Os3β-1 fusion protein, the synthesis of GA₄ (3β-hydroxylation), GA₇ (2,3-unsaturation and 3β-hydroxylation), and GA₃₄ (2β-hydroxylation) were confirmed when the substrate was GA₉ (FIG. 9), with GA₄ and GA₇ being dominant among these reaction products. Similar results were obtained when GA₂₀ was used as a substrate. Furthermore, when GA₅ and GA₄₄ were the substrate, only the corresponding 3β-hydroxylated gibberellins (GA₃ and GA₈₈) were obtained. On the other hand, when GA₅, GA₉, GA₂₀, and GA₄₄ were used as the substrate, the Os3β-2 fusion protein produced the corresponding 3β-hydroxylated gibberellins (GA₃, GA₄, GA₁, and GA₃₈) (FIG. 10).

The results revealed that the Os3β-1 gene encodes an enzyme catalyzing the nuclear reactions of 2-, 3-unsaturation and 2β-hydroxylation as well as 3β-hydroxylation. Furthermore, it became evident that the Os3β-2 gene encodes an enzyme catalyzing the 3β-hydroxylation.

INDUSTRIAL APPLICABILITY

The present invention has provided novel proteins and genes involved in the activation of plant gibberellins as well as plants whose gibberellin activity has been modified by controlling the expression of these genes. This invention enables modification of gibberellin activation in plants so as to artificially modify the plant types. Suppression of gibberellin activation in plants induces plant dwarf phenotypes due to suppression of longitudinal growth. For example, this could prevent rice plants from bending over when excessive elongation is promoted by ample fertilization. A substantial increase in crops may also be expected due to enhanced efficiency of light reception to leaves. It is also possible to improve efficiency in harvesting and breeding management. Another result of the present invention is to increase the yield of the plant as a whole by elevating the expression of genes of this invention in the plant so as to promote gibberellin activation therein. This strategy is particularly beneficial in improving the yield of feed crops as a whole. 

1. An isolated DNA encoding a protein selected from the group consisting of: (a) a DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) a DNA comprising the nucleotide sequence from position 1 to 1119 set forth in SEQ ID NO 4; and (c) a DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO:2 in which one to 10 amino acids are substituted, deleted, added, and/or inserted and wherein said protein: (i) has gibberellin 3β-hydrolase activity; (ii) conserves amino acids corresponding to amino acids from 222 to 229 and 285 to 290 in the amino acid sequence set forth in SEQ ID NO:2; and (iii) has a motif represented by Met-Trp-X-Glu-Gly-X-Thr, wherein X is any amino acid.
 2. A vector comprising the DNA according to claim
 1. 3. A transformed plant cell comprising the DNA according to claim 1 in an expressible state.
 4. A transgenic plant comprising the transformed plant cell according to claim
 3. 5. A propagative material of the transgenic plant according to claim 4, which propagative material comprises said transformed plant cell. 